Histone proteins are responsible for efficient packaging of DNA and, more importantly, the modulation of almost every chromatin-templated activity. The histone ?code? hypothesis states that the combination of a set of PTMs in histone may elicit a specific transcriptional program. In these three aims, we will develop, validate and apply a comprehensive approach, based on two flavors of mass spectrometry, to the analysis of histone modifications in several disease and model systems. Specific Aim 1. To Develop and Validate a Quantitative Triple Quadrupole (QqQ) Method to Monitor 100 Histone Modifications from 1,000 Cells using a Bottom-Up Approach. Specific Aim 2. To Develop and Validate a Quantitative Top-Down Mass Spectrometry Method to Monitor 1000 Histone Proteoforms from 10,000 Cells. Specific Aim 3. To Integrate Quantitative Measurements of Histone Modifications with Genetic Information. ? Development and Validation of a Robust Pipeline for Clinical Specimens. Using technology developed in Specific Aims 1 and 2, Specific Aim 3 integrates those mass spectrometry approaches with genetic data to provide insight into clinical samples coming from cancer patients carrying targeted mutations in epigenetic modifiers. In many cases, these mutations are thought to be the ?drivers? behind tumorigenesis. While TR&Ds 1, 2 and 3 focus on specific technical hurdles in proteomics analyses, TR&D 4 seeks to harmonize multiple, dense, diverse datasets. This TR&D project will advance the burgeoning field we refer to as ?epiproteomics? by standardizing methods and maximizing the quality and quantity of information acquired from limited and heterogeneous clinical specimens to gain insight into complex diseases.